G protein-coupled receptors transmit signals to intracellular effectors, including members of the mitogen activated protein (MAP) kinase family. We have found that receptor activation in yeast promotes ubiquitination of the MAP kinase kinase Ste7. Our hypothesis is that Ste7 ubiquitination leads to desensitization. Our objective is to determine the mechanism and function of pheromone-mediated kinase ubiquitination. There are four specific aims: Aim 1: Determine the functional consequences of Ste7 ubiquitination. Using mutants that enhance or diminish ubiquitination we will establish how ubiquitination affects pheromone signaling in vivo. Using purified ubiquitinated and unmodfied Ste7, we will establish how ubiquitination affects kinase activity in vitro. Aim 2: Determine how Ste7 ubiquitination is regulated. We will use mutants that block phosphorylation of Ste7 by upstream (Ste11) and downstream (Fus3) kinases. In addition, we will identify the Ste7 ubiquitinating enzymes and determine how their activity is regulated by pheromone. Aim 3: Determine how Ste7 degradation is regulated. Ubiquitinated Ste7 is poorly degraded in cells that lack Ubp3 (ubiquitin processing protease). We will determine if Ubp3 copurifies or otherwise interacts with the proteasome. We will identify proteins that interact with Ubp3 specifically, and determine which are required for Ste7 substrate specificity. Alternatively we will screen for factors specifically needed to degrade ubiquitinated Ste7. Aim 4: Determine how Ubp3 substrate specificity is achieved. We will sequence protein substrates that copurify with catalytically inactive Ubp3, or that accumulate in ubp3-deficient cells. Common substrate sequence elements will be evaluated for Ubp3 binding activity. These experiments will reveal how cell signaling is regulated through feedback regulation of an effector kinase, initially in yeast and eventually in mammalian cells.